Tuberculosis is an infectious disease that has been tormenting people for several thousand years, and it is estimated that ¼ of the adult population in Europe in the nineteenth century has died from tuberculosis. Thanks to improvement in living standards and the discovery of antibiotics in the twentieth century, tuberculosis has been virtually eradicated in industrialized nations by the 1950s. However, tuberculosis is still rampant as a recurrent infectious disease. In the present day, it is estimated that one in three people in the world population is infected with tubercle bacillus (latent tuberculosis). The total number of new-onset tuberculosis patients in the world is said to be 9.4 million, and it is estimated that 2 million patients among them die therefrom. Currently, approximately 95% of active tuberculosis patients live in developing countries, and 99% of people who die from tuberculosis are concentrated in developing countries.
In the 1990s, the world recognized anew that the number of tuberculosis cases has still continued to rise so as to be the greatest infectious disease centered in developing countries, and has set about to take full-fledged measures. Countermeasures against tuberculosis have been made under global cooperation, such as the deployment of Directly Observed Treatment, Short-Course (DOTS) most prominently by the World Health Organization (WHO), the establishment of “Stop Tuberculosis partnership” for advancing measures under cooperation of countries that are directly concerned, aid providing nations, and aid organization, and the founding of “The Global Fund to Fight AIDS, Tuberculosis and Malaria” as a system to provide financial support. Many of the existing technologies that are used as countermeasures against tuberculosis have been generally used for several tens of years without any improvements. Therefore, there is a pressing need to develop new technologies that are effective in developing countries.
For the diagnosis of tuberculosis, it is most important to discover tubercle bacillus infection at an early stage in order to initiate a therapy. In particular, since patients who are in a bacteria discharge state have an extremely high risk of spreading the infection to surrounding people, it is necessary to treat those patients in an isolated environment without any contact with the surrounding people. As a test for that, “Bacteriological diagnosis of tuberculosis” exists, and sputum smear test described therein is used most commonly.
The sputum smear test is a method for directly observing and examining a sputum smear sample under a microscope (direct microscopy of sputum smear sample), and was established by Robert Koch more than a century ago. Mycobacteria are identified in the clinical test sample by staining the pathogen of tuberculosis. The tuberculosis diagnostic method used today has been established by practically using the technology used by Koch. The tubercle bacillus smear test is a standard tuberculosis diagnostic method in developing countries, and is also used as a reference for evaluating performance of a new testing method.
Gene amplification method has been established as a method that has higher sensitivity than the sputum smear test. The gene amplification method is a method for amplifying a specific DNA fragment in a chain reaction manner using DNA polymerase. The principle of this method is amplification of DNA that is intended for testing, using DNA that is to be amplified, one pair of DNA primers that are complementary to sequences at both ends of the DNA, and a heat resistance DNA polymerase, and making repetitive temperature changes. Although the gene amplification method is satisfactory in term of sensitivity, it is difficult to use this method in developing countries due to operability, equipment, and cost.
As a recent global trend, because of the spread of multiple-medicament resistant tuberculosis, interest is directed toward the necessity of culture test and nucleic acid amplification test. However, among various bacteriological diagnostic methods that exist, the sputum smear test (direct microscopy of sputum) is still regarded as a central core of tuberculosis countermeasure strategies. Particularly in resource-poor developing countries in which tuberculosis is spreading, the sputum smear test is not only an effective diagnosis method, but also plays an important role as means for determining therapeutic effect.
As described above, although the sputum smear test is the central core of the basic strategy of tuberculosis control, the number of workers who perform bacteriological examination in many developing countries is absolutely insufficient, and it is said that quality of such workers is problematic. Since the effectiveness of this test depends on the skill of laboratory technicians, everyday training and quality control are essential, and these are causes for an extreme increase in overall cost. In addition, developing countries have many problems in social infrastructure required for improving laboratory environment, test instruments, testing technologies, and the like. These factors become entangled in a complicated manner, and, as a result, adversely affect quality of the test to no small extent.
Furthermore, since the sputum smear test is a test for detecting and identifying acid-fast bacilli but not a method for detecting tubercle bacilli, it is impossible to identify tubercle bacilli with the test.
Regarding tuberculosis therapy in advanced nations, therapy using a chemical agent is initiated after tubercle bacillus is identified; whereas in the DOTS program conducted in developing countries, therapy using a chemical agent is initiated when an acid-fast bacillus is detected in the sputum smear test without identifying the tubercle bacillus. Here, although chemical agents used for a tubercle bacillary infection and a nontuberculous-acid-fast bacillary infection are different, therapy is conducted for nontuberculous acid-fast bacilli basically using a chemical agent for tubercle bacilli since a highly therapeutic effect medicament for nontuberculous acid-fast bacilli has not been developed. Therefore, a patient has to bear the risk of side effects by the chemical agent. Representative examples of risks that have been pointed out include serious side effects such as decrease in liver function and loss of eyesight, and spread of medicament resistant nontuberculous acid-fast bacilli.
As described above, although there are several problems in hitherto known diagnosis of tuberculosis and therapy thereof, the sputum smear testing method developed by Koch in 1882 is still currently used for the diagnosis of tuberculosis without any large improvements. For the purpose of solving problems in developing countries and newly emerging countries regarding social infrastructure necessary for laboratory environment, test instruments, testing technologies, etc.; two measures are conceivable, i.e., “establishment of a highly sensitive and low cost testing method that enables high throughput processing of specimens collected at limited locations complete with power supply and facilities” or “establishment of Point Of Care Testing (POCT) that is highly sensitive, rapid, easy, and low cost, and that is conducted without using an instrument that requires a power supply.”
Power supplies and facilities are put into place also in developing countries and newly emerging countries if it is in a very limited area. In addition, testing centers that perform relatively advanced tests already exist in such an area, and some wealthy patients can use those services. By utilizing such testing centers, problems such as laboratory environment, test instruments, testing technologies, and human resources can be solved. In such a case, it is necessary to transport a patient or a sputum specimen from a medical-examination scene to a testing center. There are several major restrictions in transporting sputum specimens used for a nucleic acid amplification test or a culture test. In order to prevent contamination and growth of unwanted germs, specimens have to be immediately refrigerated after sputum collection to be transported. Furthermore, since it is important for a culture test to keep bacteria in a growable state, more caution is required when compared to the genetic testing. Further, since both tests cannot be performed when a sterilization operation such as heat treatment is conducted on a specimen, specimens have to be transported in a strictly controlled state. Since a system that can handle such restrictions involved in specimen transport is not established in developing countries and newly emerging countries, it is important to establish an assay capable of detection even in specimens having a sterilization operation applied thereto.
When performing POCT, it is important to enable assay at a clinical scene in a short period of time without using special facilities and instruments. The technology used mostly for POCT is immunochromatographic test (ICT). Generally, ICT is considered inferior to immunoassay methods such as ELISA in terms of sensitivity. Therefore, if the problem were to be solved with POCT, it would be important to establish an assay system capable of high sensitivity detection. Furthermore, in the case with POCT, since it is difficult to take infection prevention measures for test workers by using safety cabinets and the like used in testing centers etc., sterilization treatment for reducing risk of infection from a specimen is essential.
Therefore, it is necessary to establish an assay capable of high sensitivity detection in sterilized specimens for “establishment of a highly sensitive and low cost testing method that enables high throughput processing of specimens collected at limited locations complete with power supply and facilities” and “establishment of Point Of Care Testing (POCT) that is highly sensitive, rapid, easy, and low cost, and that is conducted without using an instrument that requires a power supply.” To do so, the selection of target antigen is important. As antigens specific for tubercle bacilli, protein antigens such as Ag85 are reported. However, since protein antigens are quickly degraded and digested in vivo, fine sensitivity can hardly be obtained therewith.
Major antigens in acid-fast bacilli include glycolipids that are major constituents of cell membranes and cell walls. Glycolipid antigens are considered to be one type of promising target antigens since they are highly stable in vivo. Among those, LAM accounts for 15% of bacterial cell components, and is an antigen that is gathering attention also in terms of its quantity. There are several reports of creating PoAb and MoAb for the purpose of detecting acid-fast bacillary LAM in live specimens (cf. Non-Patent Literatures 1 to 4 (NPL 1 to 4)). In couple of those reports, ELISA for detecting sputum or urinary lipoarabinomannan (hereinafter, also referred to as “LAM”) is used. However, basic structures of LAMs in acid-fast bacilli are almost the same except for a minute difference observed in the structure of mannose cap. In particular, in Mycobacterium avium which is a bacterium that causes acid-fast bacillary infections second most frequently only behind tubercle bacillus, the mannose cap structure is almost the same as that of tubercle bacillus (cf. Non-Patent Literature 5 (NPL 5)). Therefore, there is a desire for a monoclonal antibody capable of specifically detecting LAM of tubercle bacilli among acid-fast bacilli.